CROPNUT: increasing iron in cereals

Lead Research Organisation: University of East Anglia
Department Name: Biological Sciences

Abstract

Deficiencies in iron and zinc are global health issues, which are currently addressed by supplements and fortification programmes. In the developing world, iron supplements are an integral part of aid programmes and combatting anaemia is a major priority of the World Health Organization. Closer to home, low serum iron levels are prevalent in ~5% of females aged 11 - 64 (National Diet and Nutrition Survey 2012) correlating with low iron intake. To combat iron deficiency, all flours milled in the UK are chemically fortified with iron salts or iron powder up to 16.5 mg/kg (UK Flour Regulations 1998). The natural variation of iron in modern bread wheat is limited within the range of 6 - 13 mg/kg in white flour. Fertilization experiments have shown that extra iron is not taken up by plants. Therefore, increasing iron and zinc levels using genetic methods, known as biofortification, is the most sustainable approach to increase mineral levels in our diet which is dominated by a few staple crops.

The limited variation in iron levels in bread wheat has been attributed to a number of factors. Over centuries, crop varieties have been selected for yield, at the cost of micronutrient content. Moreover, polyploid crops such as wheat are genetically buffered: gene variants that could change a certain trait are masked by other copies of the same gene, which makes it hard to select novel traits. In addition, iron levels are tightly regulated by plants to prevent over-accumulation of this metal that is toxic in its free ionic form. And last but not least, cereals have not evolved to put large amounts of minerals into the starchy endosperm, the part of the grain that we prefer to eat.

In a very successful collaboration between the Balk and Uauy labs, we have recently found that, against expectations, iron and zinc levels in white flour from wheat or barley can be increased 3- and 2-fold, respectively. Element analysis showed that the iron levels of white flour were 16 - 17 mg/kg, similar to the legal requirement for fortification, and higher still in wholemeal flour. This was achieved by a cis-genics approach: wheat plants were genetically modified but the sequences are from wheat itself. We placed an endosperm-specific regulatory sequence in front of a wheat iron transporter. Our results show that, in principle, plants can direct much more iron and zinc to the endosperm than they do naturally. Moreover, there does not seem to be any major negative effect on growth.

While the timely overexpression of the vacuolar iron transporter works remarkably well in boosting iron and zinc levels in grain, we do not yet understand why this strategy is so successful. After all, the up-regulated gene is a simple transporter and not a regulator. If we draw an analogy to traffic flow, increasing the number of lanes on the M25 does not per se improve traffic flow. Access roads, junctions and so on all need to be widened to increase traffic and prevent congestion. Also, we found that the transporter is specific for iron and cannot transport zinc. So why are zinc levels increased?

To exploit our findings for biofortification, we will investigate the molecular and cell biological changes that underlie increased mineral transfer in the high-iron wheat line. We will also investigate what the source of iron and zinc is, for example if the plants take up more iron from the soil or whether the iron is more efficiently remobilized from other parts of the plant. We will then use this information to develop non-GM strategies to increase iron and zinc in wheat and other cereals. The bioavailability of the iron and zinc will be tested by offering digested white flour and bread to cultured intestinal cells. Taken together, these studies will greatly enhance our knowledge on nutrient transport, provide us with novel and non-GM strategies to increase the nutritional quality of wheat and give us a way to assess their impact on human nutrition.

Technical Summary

Most modern wheat varieties, although excellent providers of carbohydrates, are poor sources of mineral micronutrients. Levels of the micronutrients iron and zinc are especially low in the endosperm, which is used to make white flour. Conversely grains contain relatively high levels of the anti-nutrient phytate. Therefore, the Food Standards Agency requires all milled flour sold in the UK to be fortified with iron salts or iron powder. A much more sustainable method is biofortification, whereby plants are induced to translocate more minerals into edible parts. We have recently developed a high-iron wheat line by overexpressing a vacuolar iron transporter using an endosperm-specific promoter (Connorton et al, manuscript in preparation). The sequence are from wheat itself (cisgenic). Iron in the white flour fraction is increased 3-fold to 16 - 17 ppm, which would remove the legal requirement for fortification. However, we actually do not fully understand why this particular strategy is so successful whereas other strategies have only marginally increased iron and zinc levels. Here we propose to use the high-iron wheat line as a tool to understand how iron is transported into the grain and further distributed to the aleurone, endosperm and embryo. We will study changes in gene expression as a consequence of the increased iron flux, and map the route of iron through the different tissue and cell types using isotope studies and NanoSIMS imaging (with Dr Katie Moore, Manchester University). We will also investigate if the increased iron is due to increased uptake by the roots or increased remobilization from senescing leaves. In addition, we will investigate bioavailability of the iron for human nutrition in the white flour fractions and how this is affected by food processing, such as baking bread(with Paul Sharp, King's College London). This knowledge will be used to design non-GM approaches to increase the mineral content of cereals.

Planned Impact

The iron levels in the new high-iron wheat line are 16 - 17 mg/kg in white flour, well above the upper limit of natural variation (~ 12 mg/kg) and in line with the legal requirement for chemical fortification (16.5 mg/kg). So far, we do not see any negative impact on plant growth or yield. The work to date has been carried out in the Fielder cultivar, but this trait could now in principle be bred into modern commercial wheat varieties and remove the requirement for post-milling chemical fortification. We have contacted several potential UK stakeholders and received interested responses from the baking company Warburtons and from the National Association of British and Irish Millers (NABIM) (see letters of support).

One obstacle to more widespread acceptance is that the successful high-iron line, though not transgenic, is by definition genetically modified (GM). The approach taken was what is called cisgenic: we used a wheat promoter to change the timing and levels of expression of a wheat gene. So the sequences that were transformed are from the same species, The manipulations that are required cannot currently be achieved by non-GM methods. Although the cisgenic line is of interest to countries that do accept GM crops (e.g. India) most UK stake holders, and also the international maize and wheat improvement centre CIMMYT (see letter of support) are hesitant to use this line in their breeding programmes. We therefore seek to replicate the striking phenotype seen in this line through non-GM means.

Now that we know that a dramatic increase in iron and zinc in the endosperm of cereal grains is technically possible, it should be possible to design other, non-GM strategies to meet the same goal. For this we need to better understand what is changed in the high-iron line with regards to iron and zinc transport. We suspect that increased accumulation of iron into the vacuole of the endosperm has triggered other changes in gene expression, such as increased uptake by the roots and/or increased remobilization from senescing leaves, while lowering the saturation point of regulatory mechanisms. Though overexpressing genes in wheat through non-GM means is not currently feasible we do have a population of TILLING lines available with single nucleotide polymorphisms (SNPs) in potential genes of interest that will severely disrupt their function. These lines are not classed as GM and so through crossing with commercial wheat varieties high iron traits associated with these SNPs can be incorporated into existing breeding programmes. An alternative approach is CRISPR, which is also not classed as GM, and could be used to simultaneously knock down the function of multiple genes. The iron sensing and regulatory machinery of wheat would be excellent targets for these approaches.

Publications

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Connorton JM (2017) Iron homeostasis in plants - a brief overview. in Metallomics : integrated biometal science

 
Description We have shown that iron is redistributed in the wheat grain as a consequence of the endosperm-specific overexpression of TaVIT2. The iron content in the embryo and aleurone cell layer is lower, whereas the iron in the endosperm is increased. The phytic acid content of the endosperm has remained the same. Phytic acid is an anti-nutrient that inhibits the uptake of iron in the intestine. Using an in vitro model of intestinal cells, we have shown that the iron in the endosperm (from which white flour is milled) is more bioavailable.
Exploitation Route Too early to say
Sectors Agriculture, Food and Drink

 
Description In 2019 we applied to Defra for a licence for a small-scale field trial of the high-iron wheat line. As part of the application procedure and guidelines, we advertised this widely, including an advert in a national newspaper. This has triggered some Twitter activity from anti-GM organizations. The results at the end of the season, namely that the field trial had been successful and the plants performed as expected, was also publicized but received less attention as it coincided with the election campaign. The wheat line was obtained by Agrobacterium-mediated transformation, but we consider the line cisgenic rather than transgenic. The high-iron trait is generated by using pieces of wheat DNA in a certain combination, which does not occur normally. Thus, we did not transfer genes from other organisms to get the altered trait. Also, funding of our work is entirely from the research council and from the charity HarvestPlus. We hope that "the general public" see this as positive, and slowly change their opinion on GM crops. The PDRA on the project and Lead PI have given several talks to non-academic audiences and to professionals outside the field of crop genetics. These include: (February 2018) 2-day meeting with representatives from HarvestPlus, to discuss the pipeline to bring high-iron wheat to farmers in Bangladesh and Pakistan; (April 2018) a display of control and high-iron wheat for a meeting of the Heads of the Commonwealth at the Royal Society; (June 208) presentation of high-iron wheat at the John Innes Centre Breeders Day; (June 2018) a display and 1-to-1 interactions with the public at the Royal Norfolk Show; (September 2018) the PI gave an invited talk to a meeting on bioavailability, highlighting iron biofortification of crops; (October 2018) the PDRA gave a talk at the annual meeting of the American Association of Cereal Chemists, held in London that year; (November 2018) the PI gave a talk at the annual meeting of the Association of British and Irish Millers (NABIM) in London; (February 2019) the PI presented the high-iron wheat research to the Chief Scientific Advisor to Defra; (February 2019) the PDRA gave a tour of the field trial site during a visit by the European Landowner Organisation; (April 2019) the PI presented the high-iron wheat project to a delegation from the Indian Department of Biotechnology; (July 2019) the PI presented the research to the Executive Manager of Plants for the Future, European Technology Platform; (August 2019) the PI was interviewed on BBC breakfast TV about the GM wheat field trial; (September 2019) the Co-I was interviews on BBC Farming Today about the field trial and GM crops in general; (September 2019) the PI gave a keynote talk at the Annual Meeting of the German Plant Nutrition Society, which includes representatives of German Plant Breeding companies and the Agronomy sector; (October 2019) the PI gave an invited talk on biofortification at the 13th annual meeting of the Federation of European Nutritionist Societies in Dublin
Sector Agriculture, Food and Drink
Impact Types Societal

 
Description Alex Johnson 
Organisation University of Melbourne
Country Australia 
Sector Academic/University 
PI Contribution Dr Alex Johnson provided the rice NAS2 sequence. After cloning this into a vector together with the wheat VIT2 gene, we have used this construct to transform the wheat cultivar Fielder. We have also send the plasmid to Dr Johnson, and he has used it to transform the wheat variety Gladius in his laboratory in Melbourne.
Collaborator Contribution Dr Alex Johnson has provided us with the sequences for the NAS2 gene from rice. This gene has been inserted in a vector together with the wheat VIT2 gene under the control of an endosperm-specific promoter. We have transformed wheat with this construct and are starting to analyse the iron content of grains. The NAS2 gene encodes the enzyme nicotianamine synthase, and increased expression of this gene is expected to increase the levels of the iron chelator nicotianamine. The VIT2 protein facilitates iron storage in the vacuole. We hope that this 2-gene construct increases the iron levels even further over that seen with TaVIT2 alone.
Impact The output so far are the transformed wheat lines. If these have increased iron, they may be used for wheat breeding programmes.
Start Year 2017
 
Description Presentation at the annual NABIM meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact I presented the research on our "high-iron wheat" to British and Irish Millers. This sparked discussion on whether we should allow GM crops in Britain.
Year(s) Of Engagement Activity 2018
 
Description Publicity for GM field trial application to Defra 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact We placed a half-page advert in the Guardian on 21 January 2019, as part of the Defra application procedure for a field trial of the high-iron wheat line. We also placed information on our website. Since then, there has been quite a bit of Twitter activity, mostly from anti-GM groups. We will respond to this as part of the application procedure.
Year(s) Of Engagement Activity 2019
URL https://www.jic.ac.uk/news/application-field-trial-2019/